Replication is required for the RecA localization response to DNA damage in
            <i>Bacillus subtilis</i>

Simmons, Lyle A.; Grossman, Alan D.; Walker, Graham C.

doi:10.1073/pnas.0607123104

Cited by 56 publications

(109 citation statements)

References 55 publications

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“…In E. coli and Bacillus subtilis, RecA-GFP fusion proteins have been visualized as foci in response to DNA damage and replication of damaged DNA. 33,34) The PpRecA2-GFP foci may form in response to cpDNA damage, which might be caused in the course of the procedure for protoplast preparation and subsequent transformation.…”

Section: Discussionmentioning

confidence: 99%

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the MossPhyscomitrella patens

Inouye

Odahara

Fujita

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Section: Discussionmentioning

confidence: 99%

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the MossPhyscomitrella patens

Inouye

Odahara

Fujita

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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“…By coupling homologous recombination with DNA replication status, a cell may ensure that this requirement is met. A study using a partially functional recAgfp fusion allele integrated at the native recA locus as the only source of RecA activity in the cell showed that ongoing DNA replication was necessary for RecA-GFP to form foci in response to single-strand gaps or an I-SceI-induced DSB in vivo (387). In this study, a DSB was generated and RecA-GFP failed to organize into a focus when DNA replication initiation was blocked (387).…”

Section: Reca Recruitment Loading and Coupling To Dna Synthesismentioning

confidence: 99%

“…This could aid in vivo in the repair of DNA damage such as interstrand cross-links at replication forks, as already suggested. However, the overall roles of these proteins in repair and genome maintenance are still unclear (387). SbcE (YhaN) was recently identified as an SMC family protein in B. subtilis which has a role in DNA repair (189).…”

Section: Potential Roles For Other Smc-like Proteins In Dsb Repairmentioning

confidence: 99%

“…A study using a partially functional recAgfp fusion allele integrated at the native recA locus as the only source of RecA activity in the cell showed that ongoing DNA replication was necessary for RecA-GFP to form foci in response to single-strand gaps or an I-SceI-induced DSB in vivo (387). In this study, a DSB was generated and RecA-GFP failed to organize into a focus when DNA replication initiation was blocked (387). It is also worth noting that DNA replication has previously been shown to be necessary for SOS induction in E. coli (357).…”

Section: Reca Recruitment Loading and Coupling To Dna Synthesismentioning

confidence: 99%

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DNA Repair and Genome Maintenance in Bacillus subtilis

Lenhart

Schroeder

Walsh

et al. 2012

Microbiol Mol Biol Rev

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148

155

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SUMMARY From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.

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“…Resuspend the cells in up to 2 mL of S750 medium from the LB agar plate with single colonies or the light confluent cells to obtain an inoculum. 4. Add enough S750 medium-cell inoculum to the Erlenmeyer flask containing 10 mL S750 medium for a culture with an initial optical density measured at 600 nm (OD600) of~0.08 to 0.1.…”

Section: Protocolmentioning

confidence: 99%

Imaging Mismatch Repair and Cellular Responses to DNA Damage in <em>Bacillus subtilis</em>

Klocko

Crafton

Walsh

et al. 2010

JoVE

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Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species. ProtocolGrowing cultures of cells for microscopy 1. One or two days prior to imaging, prepare the B. subtilis strain containing the translational fusion protein you wish to visualize. Trial rounds of steps 1A and 1B will be necessary to determine which growth condition provides the best images of your strain. In most cases, cells should be imaged during exponential growth phase.2. On the morning of imaging, place 10 mL of S750 1-3 medium into a 125 mL sterile Erlenmeyer flask.3. Resuspend the cells in up to 2 mL of S750 medium from the LB agar plate with single colonies or the light confluent cells to obtain an inoculum.4. Add enough S750 medium-cell inoculum to the Erlenmeyer flask containing 10 mL S750 medium for a culture with an initial optical density measured at 600 nm (OD600) of~0.08 to 0.1. At this step it is important to have at least a 10-fold air to medium ratio. Thus, we use 10 to 12.5 mL of inoculated medium in a 125 mL Erlenmeyer flask.5. Grow each culture at 30°C until the culture reaches an OD600~0.5-0.8. Shaking water baths are preferred with revolutions per minute from 150-200. Cultures challenged with DNA damaging or mismatch inducing agents should be at an early exponential growth phase OD600 such that the cells will reach the target OD600 with the additional period of growth that is required for treatment (see Table 1 Sample Preparation1. Pipette 300 μl of cultured cells into a 1.5 mL microcentrifuge tube.2. If imaging of cellular membranes is desired, add 1:1000 dilution of FM4-64 membrane stain (Invitrogen) to each sample from a 1 m...

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Replication is required for the RecA localization response to DNA damage in Bacillus subtilis

Cited by 56 publications

References 55 publications

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the MossPhyscomitrella patens

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the MossPhyscomitrella patens

DNA Repair and Genome Maintenance in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in <em>Bacillus subtilis</em>

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